Mycobacterium tuberculosis is a major epidemic today, causing 8 million new cases and 3 million deaths yearly world-wide. New anti-tubercular drugs that act through novel mechanisms are urgently needed for more efficacious treatments in combination with existing drugs and for treating multi-drug resistant strains that emerged in recent years. The bacterial cell wall contains peptidoglycan that provides resistance from external osmotic pressure. Targeting steps of peptidoglycan biosynthesis is an attractive mechanism for new antibacterial agents. An essential step is the synthesis of D-Alanyl-D-Alanine dipeptide (D-Ala-D-Ala) that is required for the formation of peptidoglycan and for cross-linking peptidoglycan strands. The enzyme catalyzing this process is D-Alanyl-D-Alanine ligase (DDl). DDl is an excellent target for new antibiotics since synthesis of D-Ala-D-Ala is an essential process for bacterial growth and furthermore there is no human equivalent to DDl. We have applied an in silico structure based approach to identify and assay DDl ATP and substrate site binding ligands using sets of compounds that have shown activity against M. tuberculosis TB H37Rv in HTS screening and kinase binding ligands that show structural similarities to DDl. We propose to refine and validate DDl binding prediction protocols in the first half of the proposed effort and utilize these to identify and assay compounds based on known scaffolds of valid anti-tubercular targets for activity against DDl. PUBLIC HEALTH RELEVANCE: New anti-tubercular drugs that act through novel mechanisms are urgently needed for more efficacious treatments of tuberculosis and for treating multi-drug resistant strains that recently emerged. In this proposal we have apply an in silico structure based approach to identify and assay novel potent DDl binders and multi- TB target ligands.